Method for deacetylation of biopolymers

Abstract

A method for at least partial deacetylation of a biopolymer comprising acetyl groups, including: a1) providing a biopolymer including acetyl groups; a2) reacting the biopolymer including acetyl groups with hydroxylamine (NH 2 OH) or a salt thereof at a temperature of 100° C. or less for 2-200 hours to form an at least partially deacetylated biopolymer; and a3) recovering the at least partially deacetylated biopolymer.

The invention claimed is: 
     
       1. A method of preparing a hydrogel product comprising crosslinked glycosaminoglycan molecules, the method comprising:
 (a) obtaining a solution comprising an at least first glycosaminoglycan that is a partially deacetylated glycosaminoglycan with a degree of acetylation between 50% and 99% and a second glycosaminoglycan, wherein the second glycosaminoglycan is different from the at least partially deacetylated glycosaminoglycan; 
 (b) activating carboxyl groups on the at least partially deacetylated glycosaminoglycan and/or the second glycosaminoglycan with a coupling agent, to form activated glycosaminoglycans; 
 (c) crosslinking the activated glycosaminoglycans, solely with amide bonds, using a crosslinker comprising at least two amine groups, to provide crosslinked glycosaminoglycans; and 
 (d) acylating residual amine groups of the crosslinked glycosaminoglycans obtained in (c) to form acylated crosslinked glycosaminoglycans; and 
 (e) subjecting the crosslinked glycosaminoglycans provided in (c) or (d) to alkaline treatment to hydrolyze ester crosslinks formed as byproducts during the amide crosslinking in (c), 
 wherein the first and second glycosaminoglycans are not the crosslinker, and wherein the crosslinker comprises a di-, tri-, or tetra-saccharide, oligosaccharide, or polysaccharide. 
 
     
     
       2. The method according to  claim 1 , wherein the second glycosaminoglycan is at least partially deacetylated. 
     
     
       3. The method according to  claim 1 , wherein at least 1% of N-acetyl groups of the first glycosaminoglycan have been converted to free amine groups. 
     
     
       4. The method according to  claim 2 , wherein at least 1% of N-acetyl groups of the second glycosaminoglycan have been converted to free amine groups. 
     
     
       5. The method according to  claim 1 , wherein the second glycosaminoglycan is not deacetylated. 
     
     
       6. The method according to  claim 1 , wherein the first and second glycosaminoglycans are selected from hyaluronic acid, chondroitin, chondroitin sulphate, heparin sulphate, heparosan, heparin, dermatan sulphate, and keratin sulphate. 
     
     
       7. The method according to  claim 1 , wherein the first glycosaminoglycan and/or the second glycosaminoglycan is hyaluronic acid. 
     
     
       8. The method according to  claim 1 , wherein the first glycosaminoglycan and the second glycosaminoglycan are hyaluronic acids. 
     
     
       9. The method according to  claim 1 , wherein the crosslinker comprises a di, tri-, or tetra-saccharide, or combinations thereof. 
     
     
       10. The method according to  claim 1 , wherein the crosslinker comprises a disaccharide. 
     
     
       11. The method according to  claim 1 , wherein the crosslinker comprises diaminotrehalose (DATH). 
     
     
       12. The method according to  claim 1 , wherein (b) and (c) occur simultaneously. 
     
     
       13. The method according to  claim 1 , wherein (b) occurs prior to (c). 
     
     
       14. The method according to  claim 1 , wherein the hydrogel product comprises a portion of glycosaminoglycans which are not crosslinked. 
     
     
       15. The method according to  claim 1 , wherein the crosslinked glycosaminoglycan molecules comprise at least 80% by weight of the hydrogel product. 
     
     
       16. The method according to  claim 1 , wherein the first glycosaminoglycan is a partially deacetylated glycosaminoglycan with a degree of acetylation between 93% and 99%. 
     
     
       17. The method according to  claim 1 , wherein the second glycosaminoglycan obtained in a) is not altered or modified by addition or removal of functional groups. 
     
     
       18. The method according to  claim 1 , wherein the first glycosaminoglycan has a weight average molecular weight of 0.5 MDa or more. 
     
     
       19. The method according to  claim 1 , wherein the hydrogel product has a degree of amide crosslinking (CrDamide) of at least 0.5. 
     
     
       20. The method according to  claim 1 , wherein the hydrogel product has a degree of amide crosslinking (CrDamide) of at least 0.9.